Identification of Carnitine Transporter CT1 Binding Protein Lin-7 in Nervous System

_L-Carnitine is an essential component of mitochondrial fatty acid b-oxidation in the muscle and may control the acetyl moiety levels in the brain for acetylcholine synthesis. Carnitine transporter 1(CT1)is the high affinity _L-carnitine transporter whose localization was observed in the kidney, testis, liver, skeletal muscle and brain. To clarify the molecular mechanism of carnitine transport, we sought to find the interacting protein that may be related to the transport function of CT1. Using the intracellular C-terminal region of rat CT1 containing PDZ(PSD95/DLG/ZO-1)motif as bait, we performed the yeast two-hybrid screening against rat brain cDNA library. Thirty two positive clones were obtained from the 2.7×10^7 clones screened. One of them was PDZ domain-containing protein Lin-7. We found that Lin-7 interacts specifically with C-termini of CT1:deletion and mutation of the CT1 C-terminal PDZ-motif abolished the interaction with Lin-7 in the yeast two-hybrid assay. In addition, a PDZ domain within Lin-7 associates with the CT1 C-terminal. The association of CT1 with Lin-7 enhanced _L-carnitine transport activities in HEK293 cells although there is no statistical significance. Coexpression of Lin-7 and CT1 is identified in motor neurons of the spinal cord ventral horn together with Lin-2, a binding partner of Lin-7 known to assemble proteins involved in synaptic vesicle exocytosis and synaptic junctions. Therefore, Lin-7 interacts with CT1 and may regulate their subcellular distribution or function in central nervous system

Molecular Mechanism of the Urate-lowering Effects of Calcium Channel Blockers

Hyperuricemia has recently been recognized as one of the risk factors for cardiovascular diseases. Some calcium channel blockers(CCBs), commonly used in the treatment of hypertension, have been reported to decrease serum urate level. Here, we tried to elucidate the molecular mechanism of the urate-lowering effects of CCBs. We performed [^C]urate uptake in cells stably expressing human urate transporter 1, a major contributor of renal urate reabsorption and a major target of uricosuric drugs such as benzbromarone and losartan(HEK-URAT1), together with mock(HEK-mock)cells to analyze the uricosuric action of CCBs. We also measured the activity of human xanthine oxidase(XO)to determine whether CCBs have inhibitory effects on urate production. The CCBs tested were nifedipine, nilvadipine, nitrendipine, benidipine, nisoldipine, nicardipine, efonidipine, amlodipine, azelnidipine, verapamil and diltiazem. We found for the first time that at least seven CCBs in the dihydropyridine subgroup interacted with URAT1-mediated urate uptake in HEK-URAT1 cells. Among these CCBs, nifedipine, nilvadipine and nitrendipine strongly inhibited URAT1-mediated urate uptake. Their IC_s were 15.8, 0.018 and 0.40?μM, respectively. In contrast, urate production mediated by XO was weakly inhibited by nifedipine and nisoldipine. In summary, URAT1 interacted with various CCBs differently, whereas XO, a major enzyme for urate production in the liver, did not interact with most of CCBs. Although CCBs were not excreted from the urine basically, their urate-lowering effects may be associated with the inhibition of renal urate reabsorption mediated by renal urate transporters such as URAT1 with their metabolites, and the results for structure-activity information in this study will provide a clue for developing new uricosuric drugs targeting URAT1

ヒト ジンゾウ ニョウサン トランス ポーター URAT 1 ト スイヨウ セイ ヨード ケイ ゾウエイザイ IODIPAMIDE ノ ソウゴ サヨウ

降圧薬などいくつかの薬剤は本来の薬理作用とは別に尿酸降下作用を持つものがあり,水溶性ヨード系造影剤もその一つでiodipamideやdiatrizoateでの尿酸排泄亢進が報告されていた.長らく不明のままであった腎尿酸輸送機構の分子実体は2002年の腎尿細管尿酸トランスポーターURAT1(Urate Transporter 1)の分子同定によりその理解が飛躍的に進んだ.本研究ではURAT1と水溶性造影剤のiodipamideおよびdiatrizoateの相互作用を検討することで,その尿酸排泄促進作用の分子機序の解明を目的とする.URAT1の尿酸輸送活性の測定にはURAT1安定発現HEK293細胞(HEK-URAT1)細胞を用いた.IodipamideはHEK-URAT1細胞でのRI標識尿酸取込みを著明に阻害した(IC_:1.19±0.08?μM)のに対し,diatrizoateは1?mMまでの範囲では50%以上の阻害作用を示さなかった.1?mMまでのiodipamideはHEK-URAT1細胞の生存率に影響を与えなかった.IodipamideによるURAT1媒介尿酸輸送への阻害作用のキネティクス解析の結果,その阻害は競合阻害であり,阻害定数Ki値は11.03?μMであった.以上より,iodipamideは尿酸トランスポーターURAT1と相互作用をすることを初めて確認できた.このことからiodipamideは細胞外からURAT1の尿酸結合部位に結合し,競合して阻害を行うことで,腎尿細管の経上皮性尿酸再吸収を抑制し,ひいては血清尿酸値を低下させるものと考えられた.Drug-induced hypouricemia has been found in several drugs such as probenecid, benzbromarone and angiotensin II receptor blocker(ARB)losartan. Xray contrast agents such as iodipamide and diatrizoate, used for the intravenous cholangiography and excretory urography, were reported to have uricosuric effct beside their original action. After the molecular identification of renal apical urate transporter URAT1 as an entrance of urate into the epithelial cells of proximal tubules, this protein is thought to be major determinant for renal reabsorption of urate that affect the blood urate levels in human. The purpose of this study is to examine whether iodipamide and diatrizoate act on URAT 1 . In URAT 1 -stably expressing HEK293(HEK-URAT1)cells, iodipamide inhibited [^C] urate uptake dose-dependently(IC_ , 1.19±0.08 μM), while diatrizoate did not. Up to the concentration of 1 mM, iodipamide incubation for 24 hr did not affect the viability of HEK293-URAT1 cells. Lineweaver-Burk plot of the kinetic analysis by URAT1-mediated urate uptake with or without iodipamide indicated that its interaction occurs in a competitive manner(Ki:11.03 μM). These results suggest that uricosuric effect of iodipamide can be explained by the interaction of iodipamide with urate-binding site of URAT1, and the inhibition of urate reabsorption from extracellular side by iodipamide causes uricosuria leading to induce hypouricemia

Involvement of K^+ Channels and Na^+, K^+-ATPase in Relaxant Actions of Selective Phosphodiesterase 3 Inhibitors on Airway and Vascular Smooth Muscles Isolated from Guinea-pigs

Milrinone or olprinone, a selective phosphodiesterase (PDE) 3 inhibitor, has relaxant actions on smooth muscles in addition to positive inotropic actions. The exact mechanism on vasodilating and bronchodilating actions of milrinone or olprinone has not been elucidated. In the present experiments, relaxant responses to PDE3 inhibitors were examined on the precontracted airway or pulmonary artery smooth muscle preparations to clarify their mechanism. Both milrinone and olprinone relaxed the airway smooth muscle preparation or the pulmonary artery preparation isolated from guinea-pigs in a concentration-dependent manner. In the airway smooth muscle, these relaxations were markedly blocked by iberiotoxin (a blocker of large conductance Ca^-activated K^+ channels). On the other hand, in the main pulmonary artery, the milrinone- and olprinone-induced relaxations were significantly blocked by iberiotoxin, and were more strongly blocked by ouabain (an inhibitor of Na^+, K^+-ATPase). In the right/left (R/L) pulmonary artery, ouabain also strongly blocked relaxant responses to milrinone and olprinone, but iberiotoxin did not modify these relaxations. Similar observations were seen on the bucladesine (a cyclic AMP mimic agent)-induced relaxation. In conclusion, milrinone and olprinone cause concentration-dependent relaxations of the isolated airway and pulmonary artery smooth muscles via an increase in intracellular cyclic AMP (cAMP). In the airway smooth muscle, large conductance Ca^-activated K^+ (BK_) channels seem to play a crucial role for these relaxations. Relaxations of the main pulmonary artery induced by milrinone and olprinone are mediated predominantly by activation of Na^+, K^+-ATPase, and partly through BK_ channels. In the R/L pulmonary artery, vasorelaxant effects of milrinone and olprinone are more likely mediated by activation of Na^+, K^+-ATPase, but not BK_ channels

Research Records and Possibilities of the Department of Pharmacology Physiology in Kerman University of Medical Sciences, Iran

Background: The publication of information and possibilities of a university department and the experiences of its faculty members make others aware of these issues and can be regarded as one of the methods of publishing and teaching science. It seems that the report of research experiences, capabilities, and achievements in Department of Pharmacology Physiology, Kerman University of Medical Sciences, Iran, make it easier for further researches of other researchers. Methods: Data were obtained via observing, searching in valuable scientific databases and group archives, and asking the department manager, faculty members, and experts in Department of Pharmacology Physiology, as well as gathering the information in research centers of the university. Results: Department of Physiology and Pharmacology was the first group that initiated postgraduate and doctoral degrees’ courses at Kerman University of Medical Sciences. The annual per capita of paper production in this group was close to 7 with 10 faculty members. Three faculty members were among the first 15 of the university in terms of the H-index. The country, provincial, university, and faculty rankings achieved by the department, variety in research projects, and the collaborative studies in the university were the features of this department. The first and second research centers of the university in terms of history and rank were managed by faculty members of this department. Conclusion: Department of Pharmacology Physiology is one of the successful and high history departments in Kerman University of Medical Sciences. Other researchers can use the experience of these faculty members, and department facilities for their advancement. Keywords Physiology; research; Equipment; Department; Pharmacy facult